Abstract

Recombinant (r) Mycobacterium bovis strains were constructed that secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of the hly gene, respectively. Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain. By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages. In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly. Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered. Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.

Footnotes

↵‡ To whom reprint requests should be addressed. e-mail: hess{at}mpiib-berlin.mpg.de.

This paper was submitted directly (Track II) to the Proceedings Office.

Edited by Gustav J. V. Nossal, University of Melbourne, Victoria, Australia, and approved February 9, 1998 (received for review October 20, 1997)

Abstract

Recombinant (r) Mycobacterium bovis strains were constructed that secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of the hly gene, respectively. Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain. By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages. In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly. Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered. Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.

Footnotes

↵‡ To whom reprint requests should be addressed. e-mail: hess{at}mpiib-berlin.mpg.de.

This paper was submitted directly (Track II) to the Proceedings Office.

A study examines trends in global fishing fleets and finds that by 2015, 68% of the global fishing fleet became motorized, and that the overall number of fleet vessels increased to 3.7 million, despite a consistent decrease in the catch per unit of effort.

A method to determine gender from fingerprints suggests pottery making was not a primarily female activity in ancient Puebloan society, challenging previous assumptions about gendered divisions of labor in ancient societies.